The need for catalysts that operate outside of their native context or which catalyze reactions that are not represented in nature has resulted in the development of “enzyme engineering” technology. The usual route taken in enzyme engineering has been a “rational design” approach, relying upon the understanding of natural enzymes to aid in the construction of new enzymes. Unfortunately, the state of proficiency in the areas of protein structure and chemistry is insufficient to make the generation of novel biological catalysts routine.
Recently, a different approach for developing novel catalysts has been applied. This method involves the construction of a heterogeneous pool of macromolecules and the application of an in vitro selection procedure to isolate molecules from the pool that catalyze the desired reaction. Selecting catalysts from a pool of macromolecules is not dependent on a comprehensive understanding of their structural and chemical properties. Accordingly, this process has been dubbed “irrational design” (Brenner et al., Proc. Natl. Acad. Sci. USA, 89:5381-5383, 1992).
Most efforts to date involving the rational design of enzymatic RNA molecules or ribozymes have not led to molecules with fundamentally new or improved catalytic function. However, the application of irrational design methods via a process we have described as “directed molecular evolution” or “in vitro evolution”, which is patterned after Darwinian evolution of organisms in nature, has the potential to lead to the production of DNA molecules that have desirable functional characteristics.
This technique has been applied with varying degrees of success to RNA molecules in solution (see, e.g., Mills et al, Proc. Natl. Acad. Sci. USA, 58:217, 1967; Green et al, Nature, 347:406, 1990; Chowrira et al, Nature, 354:320, 1991; Joyce, Gene, 82:83, 1989; Beaudry et al, Science, 257:635-641, 1992; Robertson et al, Nature, 344:467, 1990), as well as to RNAs bound to a ligand that is attached to a solid support (Tuerk et al, Science, 249:505, 1990; Ellington et al, Nature, 346:818, 1990). It has also been applied to peptides attached directly to a solid support (Lam et al, Nature, 354:82, 1991); and to peptide epitopes expressed within a viral coat protein (Scott et al, Science, 249:386, 1990; Devlin et al, Science, 249:249, 1990; Cwirla et al, Proc. Natl. Acad. Sci. USA, 87:6378, 1990).
It has been more than a decade since the discovery of catalytic RNA (Kruger et al, Cell, 31:147-157, 1982; Guerrier-Takada et al, Cell, 35:849-857, 1983). The list of known naturally-occurring ribozymes continues to grow (see Cech, in The RNA World, Gesteland & Atkins (eds.), pp. 239-269, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1993); Pyle, Science, 261:709-714, 1993; Symons, Curr. Opin. Struct. Biol., 4:322-330, 1994) and, in recent years, has been augmented by synthetic ribozymes obtained through in vitro evolution. (See Joyce, Curr. Opin. Struct. Biol., 4:331-336, 1994; Breaker et al, Trends Biotech., 12:268-275, 1994; Chapman et al, Curr. Opin. Struct. Biol., 4:618-622, 1994).
It seems reasonable to assume that DNA can have catalytic activity as well, considering that it contains most of the same functional groups as RNA. However, with the exception of certain viral genomes and replication intermediates, nearly all of the DNA in biological organisms occurs as a complete duplex, precluding it from adopting a complex secondary and tertiary structure. Thus it is not surprising that DNA enzymes have not been found in nature.
Until the advent of the present invention, the design, synthesis and use of catalytic DNA molecules with nucleotide-cleaving capabilities has not been disclosed or demonstrated. Therefore, the discoveries and inventions disclosed herein are particularly significant, in that they highlight the potential of in vitro evolution as a means of designing increasingly more efficient catalytic molecules, including enzymatic DNA molecules that cleave other nucleic acids, particularly RNA.